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pH and temperature are two important characteristics in cells and the environment. These, not only in the well-done regulation of cell functions but also in diagnosis and treatment, have a key role. Protein-protected bimetallic nanoclusters are abundantly used in the building of biosensors. However, insulin-stabilized Au-Ag nanoclusters with dual intrinsic emission have not been investigated yet. In this work, Dual emissive insulin templated Au-Ag nanocluster (Ins(Au/Ag)NCs) were first synthesized in a simple and green one-put manner. The two emission wavelengths of, as-prepared NCs centered at 410 and 630 nm, excited in one excitation wavelength (330 nm). These two emission peaks were assigned to the di-Tyrosine cross-linked formation and bimetallic nanoclusters respectively. Further analysis displayed that each emission band of Ins(Au/Ag)NCs responded to one variable whilst another peak remained constant; For blue and red emission wavelengths, pH dependency and thermo-responsibility were observed respectively. As-prepared nanoprobe with the intrinsic dual emissive feature was used for ratiometric determination of these parameters, each with a discrete response from another. The linear range of 6.0-9.0 for pH and 1 to 71 °C for temperature was obtained, which comprises the physiological range of pH and temperature and afforded intracellular sensing and imaging capability. As-prepared NCs probe show excellent biocompatibility and cell membrane permeability, and so were successfully applied as direct ratiometric pH and temperature probes in HeLa and HFF cells. More interestingly, this dual emissive nanoprobe is capable of distinguishing cancer cells from normal ones., Competing Interests: Declaration of competing interest The authors whose names are listed immediately below certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Breast cancer detection at an early stage significantly increases the chances of successful treatment and survival. This study presents an electrochemical biosensor for detecting breast cancer cells, utilizing silver nanoclusters encapsulated by hemoglobin and Cu (II)-porphyrin-metal organic framework (BioMOF) in a graphene-incorporated nanohybrid probe. This Hb-AgNCs@MOF-G probe demonstrates high electrochemical activity, superior dispersity, porosity, and a large surface area for effective functionalization. Using a green ultrasonic-assisted stirring method, we fabricate ultra-small 5 nm particles that readily immobilize on a glassy carbon electrode, generating a detection signal when interacting with ferricyanide/ferrocyanide redox probes. The resulting immunosensor detects as few as 2 cells/mL using Electrochemical Impedance Spectroscopy (EIS) "signal on" and 16 cells/mL via Square Wave Voltammetry (SWV) "signal off", within a broad range of cell concentrations (10 2 -5 × 10 4 cells/mL). Our designed sensor shows improved selectivity (5- to 16-fold) and robust detection in human blood with a recovery efficiency between 94.8-106% (EIS method) and 95.4-111% (SWV method). This sensor could streamline early cancer diagnosis and monitor patient treatment without requiring labelling or signal amplification. As a pioneering endeavor, we've utilized integrated porous MOFs with Hb-encapsulated silver nanoclusters in cancer detection, where these components collectively enhance the overall functionality., (© 2023. The Author(s).)

Early detection of breast cancer, the first main cause of death in women, with robust assay platforms using appropriate biomarkers is of great importance for diagnosis and follow-up of the disease progression. This paper introduces an extra selective and sensitive label-free aptasensor for the screening of BRCA1 gene biomarker by taking advantage of a gate modified with aptamer and molecularly imprinted polymer hybrid (MIP) as a new synthetic receptor film coupled with an electrolyte-gated molybdenum disulfide (MoS 2 ) field-effect transistor (FET). The Au gate surface of FET was modified with insulin stabilized bimetallic Ag-Au@nanoclusters (Ag-Au@InsNCs), after which, the immobilization of the hybridized aptamer and o-phenylenediamine was electropolymerized to form an aptamer-MIP hybrid receptor. The output characteristics of Apta-MIP hybrid modified Au gate MoS 2 FET device were followed as a result of change in electrical double layer capacitance of electrolye-gate interface. The magnitude of decrease in the drain current showed a linear response over a wide concentration range of 10 aM to 1 nM of BRCA1 ssDNA with a sensitivity as high as 0.4851 μA/decade of concentration and a limit of detection (LOD) of 3.0 aM while very low responses observed for non-imprinted polymer. The devised aptasensor not only was capable to the discrimination of the complementary versus one-base mismatch BRCA1 ssDNA sequence, but also it could detect the complementary BRCA1 ssDNA in spiked human serum samples over a wide concentration range of 10 aM to 1.0 nM with a low LOD of 6.4 aM and a high sensitivity 0.3718 μA/decade., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

We report the development of a label-, antibody-, enzyme-, and amplification-free ratiometric fluorescent biosensor for low-cost and rapid (less than 12 min) diagnosis of COVID-19 from isolated RNA samples. The biosensor is designed on the basis of cytosine-modified antisense oligonucleotides specific for either N gene or RdRP gene that can form silver nanoclusters (AgNCs) with both green and red emission on an oligonucleotide via a one-step synthesis process. The presence of the target RNA sequence of SARS-CoV-2 causes a dual-emission ratiometric signal transduction, resulting in a limit of detection of 0.30 to 10.0 nM and appropriate linear ranges with no need for any further amplification, fluorophore, or design with a special DNA fragment. With this strategy, five different ratiometric fluorescent probes are designed, and how the T/C ratio, the length of the stem region, and the number of cytosines in the loop structure and at the 3' end of the cluster-stabilizing template can affect the biosensor sensitivity is investigated. Furthermore, the effect of graphene oxide (GO) on the ratiometric behavior of nanoclusters is demonstrated and the concentration-/time-dependent new competitive mechanism between aggregation-caused quenching (ACQ) and aggregation-induced emission enhancement (AIE) for the developed ssDNA-AgNCs/GO nanohybrids is proposed. Finally, the performance of the designed ratiometric biosensor has been validated using the RNA extract obtained from more than 150 clinical samples, and the results have been confirmed by the FDA-approved reverse transcription-polymerase chain reaction (RT-PCR) diagnostic method. The diagnostic sensitivity and specificity of the best probe is more than >90%, with an area under the receiver operating characteristic (ROC) curve of 0.978.

This review discusses recent advances and the reported strategies over the last ten years on the use of carbon-based quantum dots (QDs), including carbon dots (CDs), graphene quantum dots (GQDs), and polymer dots (PDs) in the design of fluorescence imaging and biosensing system for early diagnosis of cancers. Besides, this study comprehensively reports the latest developments in these years in the fluorescence imaging (FI) area with special attention to carbon-based QDs that take advantage of the excellent properties offered by these zero-dimensional (0D) nanomaterials as fluorescent tags. The most remarkable advantages of these carbon nanomaterials in the development of fluorescence sensing and imaging strategies compared to the conventional dyes arise from sharp emission spectra, long photostability, low-cost synthesis, reliability, reproducibility, high fluorescent intensity, and high surface functional groups such as carboxyl and amide, which impart better solubility in many solvents and aqueous media and facilitate their easy functionalization with biological species. The final section discusses the main challenges to be met to take full advantage of these properties in fluorescence bio-sensing and imaging as well as the possible future trends in this field based on the great advances that have occurred in recent years., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Recently, there has been a growing demand to develop portable devices for the fast detection of contaminants in food safety, healthcare, and environmental fields. Herein, two biosensing methods were designed by the use of nicotinamide adenine dinucleotide phosphate (NAD(P)H)-dependent TetX2 enzyme activity and thionine as an excellent electrochemical and colorimetric mediator/probe to monitor tetracycline (TC) in milk. The nanoporous glassy carbon electrode (NPGCE) modified with polythionine was first prepared by electrochemically and then TetX2 was immobilized onto the NPGCE using polyethyleneimine. The prepared biosensor provided a high electrocatalytic response toward NAD(P)H by significantly reducing its overpotential. The proposed biosensor exhibited a detection limit of 40 nM with a linear range of 0.1-0.8 μM for TC determination. Besides, the thionine probe was used to develop a novel colorimetric assay using a simple enzymatic color reaction within a few minutes. The limit of detection for TC was experimentally achieved as 60 nM, which was lower than the safety levels established by the World Health Organization (225 nM). The correlation between change in the color of the solution and the concentration of TC was used for quality control of milk samples, as confirmed by the standard high-performance liquid chromatography method. The results show the great potential of the proposed assays as portable instruments for on-site TC measurements., (© 2020 International Union of Biochemistry and Molecular Biology, Inc.)

In this paper, a non-enzymatic glucose sensor was developed based on a g-C 3 N 4 /NiO/CuO nanocomposite immobilized on a glassy carbon electrode (GCE). Field emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), and Brunauer-Emmett-Teller (BET) were utilized for the characterization of the synthesized g-C 3 N 4 /NiO/CuO nanocomposite. The electrocatalytic activity of the nanocomposite was investigated by cyclic voltammetry, and the amperometric technique was applied for monitoring glucose. The g-C 3 N 4 /NiO/CuO/GCE exhibited better electrocatalytic performance than g-C 3 N 4 /GCE, g-C 3 N 4 /CuO/GCE and g-C 3 N 4 /NiO/GCE. Under optimized conditions, the proposed sensor offered a linearity ranging from 0.4 μM to 8.5 mM with a detection limit of 0.1 μM and a sensitivity of 362.12 μA mM -1  cm -2 . The constructed sensor displayed favorable reproducibility, outstanding selectivity, and long-term performance. These results reveal that the sensor is a promising candidate for blood glucose sensing., (Copyright © 2020 Elsevier Inc. All rights reserved.)

For the first time, we have successfully synthesized stable graphene nanosheets from graphite powder through sonication in the hemoglobin-capped gold nanoclusters (Hb@AuNCs) solution for biosensing application. This approach, as a simple method for the exfoliation and fragmentation of graphite in a nanocluster solution, enabled us to produce stable aqueous graphene dispersions at low cost and without the need for hazardous chemicals or tedious experimental procedures. In this method, Hb@AuNCs were used not only as stabilizing agent of graphene through non-covalent bonding, but also as dispersing agent of few-layer graphene nanosheets. The Hb@AuNCs stabilized graphene (Hb@AuNCs-G) was characterized by high resolution transmission electron microscopy (HRTEM), zeta-sizer and Raman spectroscopy. Then, the graphene nanosheets were applied as a novel versatile electrochemical platform for ultrasensitive biosensing of short DNA species of chronic myelogenous leukemia (CML) based on the "signal off" and "signal on" strategies. For this purpose, a single strand DNA (ssDNA) was immobilized on the Hb@AuNCs-G/AuNPs modified electrode surface and acted as the biorecognition element. Methylene blue (MB), as the signaling probe, was then intercalated into the ssDNA. The intercalated MB was liberated upon interaction with the synthetic complementary DNA (cDNA, target), thereby resulting in the apparent reduction of MB redox signal. This designed "signal off" sensing system enabled the voltammetric determination of the target cDNA over a dynamic linear range (DLR) of 0.1 fM to 10 pM with a limit of detection (LOD) of 0.037 fM. In the "signal on" strategy, the response to the cDNA was detected by monitoring the change in the electron transfer resistance (R ct ) using the ferro/ferricyanide system as a redox probe. The charge transfer resistance of the probe was found to increase linearly with increasing concentration of target cDNA in the range of 0.1 fM-10 pM with a limit of detection of 0.030 fM. Finally, the selectivity and feasibility of genosensor was evaluated by the analysis of derived nucleotides from mismatched sequences and the clinical samples of patients with leukemia as real samples, respectively., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors gratefully acknowledge the support of this work by Iran National Science Foundation (INSF) (grant number: 98004333) and the Research Council of Razi University., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The recent rapid advances in the synthesis, functionalization and application of nanomaterials have enabled scientists to develop metal nanoclusters (MNCs) stabilized with a variety of scaffolds/protecting ligands including thiols, polymers, proteins, dendrimers and nucleic acids. Considering the unique optical, electronic and physical properties of MNCs, they have been successfully used for the tumor marker biosensing assays. In recent years, the ultrasensitive and accurate detection of tumor markers has been of critical importance for the screening or diagnosis of cancers at their early stages. Nanoclusters have revolutionized the design of biosensors and provided an opportunity for the selective and sensitive determination of tumor markers. Here, we review the synthesis, stabilization and promising applications of fluorescent MNCs, with particular focus on their potential for designing tumor marker biosensors. Finally, the current challenges and future perspectives on the emerging MNC-based biosensors are highlighted as well. Our intended audiences are the broader scientific communities interested in the nanomaterial-based biosensors, and our review paper will, hopefully, open up new horizons for those scientists who manipulae the biological properties of nanoclusters. This review is based on publications available up to January 2020., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In this study, a simple and cost-effective electrochemical DNA biosensor was developed for sensitive detection of mycobacterium tuberculosis (TB). Nanocomposite of zinc oxide (ZnO) and gold nanoparticles (AuNPs) was used as a platform for immobilizing thiolated TB DNA (probe DNA). ZnO was electrodeposited on a glassy carbon electrode by potentiostat electrolysis of Zn (NO 3 ) 2 solution at -1.0 V (vs. Ag/AgCl), then AuNPs were loaded as the second layer at -0.4 V from HAuCl 4 solution. Thiolated probe DNA was then covalently attached to AuNPs. Anodic peak current of Fe (CN) 6 3-/4- was followed in hybridization experiments and a linear calibration curve was obtained in concentration range of 2.5-250 pM and limit of detection (LOD) of 1.8 pM for target DNA. The label-free TB biosensor exhibited high selectivity, suitable stability, and reproducibility., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Human immunodeficiency virus (HIV) is a lentivirus that leads to acquired immunodeficiency syndrome (AIDS). With increasing awareness of AIDS emerging as a global public health threat, different HIV testing kits have been developed to detect antibodies (Ab) directed toward different parts of HIV. A great limitation of these tests is that they can not detect HIV antibodies during early virus infection. Therefore, to overcome this challenge, a wide range of biosensors have been developed for early diagnosis of HIV infection. A significant amount of these studies have been focused on the application of nanomaterials for improving the sensitivity and accuracy of the sensing methods. Following an introduction into this field, a first section of this review covers the synthesis and applicability of such nanomaterials as metal nanoparticles (NPs), quantum dots (QDs), carbon-based nanomaterials and metal nanoclusters (NCs). A second larger section covers the latest developments concerning nanomaterial-based biosensors for HIV diagnosis, with paying a special attention to the determination of CD4 + cells as a hall mark of HIV infection, HIV gene, HIV p24 core protein, HIV p17 peptide, HIV-1 virus-like particles (VLPs) and HIV related enzymes, particularly those that are passed on from the virus to the CD 4+ T lymphocytes and are necessary for viral reproduction within the host cell. These studies are described in detail along with their diverse principles/mechanisms (e.g. electrochemistry, fluorescence, electromagnetic-piezoelectric, surface plasmon resonance (SPR), surface enhanced Raman spectroscopy (SERS) and colorimetry). Despite the significant progress in HIV biosensing in the last years, there is a great need for the development of point-of-care (POC) technologies which are affordable, robust, easy to use, portable, and possessing sufficient quantitative accuracy to enable clinical decision making. In the final section, the focus is on the portable sensing devices as a new standard of POC and personalized diagnostics., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Four fluorescent DNA-stabilized fluorescent silver nanoclusters (DNA-AgNCs) were designed and synthesized with differences in lengths of cytosine-rich DNA strand (as the stabilizing agent) and target-specific strand DNA aptamers for adenosine triphosphate (ATP) and cytochrome c (Cyt c ). After their nanohybrid formation with graphene oxide (GO), it was unexpectedly found that, depending on the composition of the base and length of the strand DNA aptamer, the fluorescence intensity of three of the nanohybrids significantly enhanced. Our experimental observations and quantum mechanical calculations provided an insight into the mechanisms underlying the behavior of DNA-AgNCs/GO nanohybrids. The enhanced fluorescence was found to be attributed to the aggregation-induced emission enhancement (AIE) characteristic of the DNA-AgNCs adsorbed on the GO surface, as confirmed evidently by both fluorescence and transmission electron microscopies. The AIE is a result of hardness and oxidation properties of GO, which lead to enhanced argenophilic interaction and thus to increased Ag(I)-DNA complex shell aggregation. Consequently, two of the DNA-AgNCs/GO nanohybrids were successfully extended to construct highly selective, sensitive, label-free, and simple aptasensors for biosensing of ATP (LOD = 0.42 nM) and Cyt c (LOD = 2.3 nM) in lysed Escherichia coli DH5 α cells and mouse embryonic stem cells, respectively. These fundamental findings are expected to significantly influence the designing and engineering of new AgNCs/GO-based AIE biosensors.

We report on facile synthesis and characterization of phosphate-functionalized polymer dots (PDs) by doping tributyl phosphate (TBP) in a semiconducting polymer poly[9,9-dioctylfluorenyl-2,7-diyl)-co-1,4-benzo-{2,10-3}-thiadiazole)] (PFBT). Then, the prepared TBP@PFBT PDs were used to develop a very high sensitive probe for detection Fe 3+ , Cu 2+ ions and Cytochrome c based on aggregation induced fluorescence off mechanism. The PDs exhibited a linear dynamic range for Fe 3+ from 0.1 to 2 nM with a detection limit of 30 pM and for Cu 2+ from 2.0 to 50.0 nM with a detection limit of 0.35 nM. Meanwhile, this probe showed a linear dynamic range for Cyt c from 175 to 1750 pM with a detection limit of 32.7 pM. The TBP@PFBT PDs is a simple, one-step, fast, non-invasive, label-free, and inexpensive probe that is capable of online apoptosis monitoring response to drugs with an ever-present opportunity to contribute in a variety of in-vitro and in-vivo biological applications. We also obtained sharp, specific 2D and 3D imaging results for early stage apoptosis in breast cancer cells. Moreover, this technique possesses the advantage of rapid determination of Fe 3+ ion in biological or environmental samples. Importantly, this label-free assay provides short determination time of only a few min, easy operation and very low LOD allowing 100-4000 times increased in sensitivity over previously reported probes, together with high selectivity without need to using biorecognition elements like enzymes, antibodys and/or aptamers. Such excellent features make the TBP@PFBT PDs an excellent probe for successful apoptosis imaging in live cells., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In this work, an electrochemical aptasensor was developed for sensitive detection of MUC1 based on metal-organic framework-reduced graphene oxide nanocomposite (Cu-MOF-RGO). Cu- MOF-RGO appeared to be suitable as a platform for immobilization of MUC1 aptamer, and also as an electrochemical probe, which exhibited well-defined peaks with good stability and reproducibility. Cu-MOF-graphene oxide (Cu-MOF-GO) nanocomposite was prepared and cast on the electrode surface, then in order to increase the conductivity of the electrode, GO was electrochemically reduced to RGO. In the presence of MUC1, the peak current of Cu in the nanocomposite decreased, which could be explained based on the formation of MUC1-aptamer complexes on the electrode, and consequence blocking the electron transfer of Cu at the electrode surface. Under optimum experimental conditions, a linear calibration curve was obtained by differential pulse voltammetry in the concentration range of 0.1 pM-10 nM (25 pg mL -1 - 2500 ng mL -1 ) with a limit of detection (LOD) of 0.033 pM (7.5 pg mL -1 ) of MUC1. The proposed aptasensor offers acceptable selectivity, stability, and reproducibility in the determination of MUC1 spiked to human blood serum samples., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In this study, a simple and lable-free voltammetric immunosensor was successfully developed for the ultrasensitive detection of prostate specific antigen (PSA). To do this, multiwalled carbon nanotube (MWCNT)/L-histidine functionalized reduced graphene oxide (His-rGO) was demonstrated as a bifunctional nanoplatform for covalently attaching thionine redox indicator and anti-PSA antibody (Ab). The MWCNT enhanced electrical conductivity and facilitated the electron transfer between thionine and the glassy carbon electrode. While, the presence of anti-PSA antibody blocked the electron transfer of thionine and decreased redox signals. The principle response of proposed immunosensor was based on the selective interaction of PSA with thionine-NH 2 -GO-COOH-Ab. This selective interaction led to further decrease of response current of attached electrochemical probe. The liner calibration curve for tumor marker determination was 10 fg mL -1 -20 ng mL -1 (R 2  = 0.996). Under optimized conditions, the immunosensor was able to selectively detect PSA with a limit of detection (LOD) of 2.8 fg mL -1 at 3σ. The relative standard deviations (RSDs) for single-electrode repeatability and electrode-to-electrode reproducibility were less than 2.9% and 5.7% (n = 5), respectively. Furthermore, the as-proposed immunosensor showed excellent performance in detection of PSA in the human serum and saliva samples, which implies that the current strategy has a promising feature for the clinical assessment of tumor marker status in patients with prostate cancer., (Published by Elsevier B.V.)

This study introduces a signal amplification strategy rely on incorporating a specific polymer film between two typical nanostructured layers, aiming to improve the electrical properties of the platform to be able to transduce small binding event through sub-femtomolar detection of HIV-1 gene at the surface of the constructed biosensing device. The proposed composite was arrayed based on a conductive layer consist of p-aminobenzoic acid (PABA) sandwiched between the electrochemically reduced graphene oxide (ERGO) as the sub-layer, and the gold nanoparticles (AuNPs) as the interfacial layer. We computationally explored that how the use of such design enables the platform to transduce small changes in the interfacial properties of the biosensor, caused by low concentrations of HIV-1 gene, without needing any amplification strategy. Furthermore, it was found that the loin PABA conductive polymer sandwiched between two nanostructure layers play an artwork-ensemble role, which resulted in a good signal repeatability and stability during the relatively long successive incubation and detection procedures. The justification of using such an array of conductive layers was established on the attaining extra low-level of detection limit. The observed performance for probe-DNA immobilized on glassy carbon electrode (GC) modified with ERGO/PABA/AuNPs compared to the GC electrode modified with ERGO/AuNPs inspired us to perform computational calculations, a hybrid of ab-initio and semi-empirical quantum mechanics methods, to discover its probable molecular-scale reasons. A rapid single frequency impedance measurement (SFIM) was also employed to remarkably reduce the measurement time and diminish the probable nonspecific impedance changes. The proposed biosensor was used to evaluate the DNA target over an extremely wide concentration range from 0.1 fM to 10 nM, with a detection limit of 37 aM (S/N = 3)., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Early diagnosis of cardiovascular disease (CVD) is critically important for successful treatment and recovery of patients. At present, detection of CVD at early stages of its progression becomes a major issue for world health. The nanoscale electrochemical biosensors exhibit diverse outstanding properties, rendering them extremely suitable for the determination of CVD biomarkers at very low concentrations in biological fluids. The unique advantages offered by electrochemical biosensors in terms of sensitivity and stability imparted by nanostructuring the electrode surface together with high affinity and selectivity of bioreceptors have led to the development of new electrochemical biosensing strategies that have introduced as interesting alternatives to conventional methodologies for clinical diagnostics of CVD. This review provides an updated overview of selected examples during the period 2005-2018 involving electrochemical biosensing approaches and signal amplification strategies based on nanomaterials, which have been applied for determination of CVD biomarkers. The studied CVD biomarkers include AXL receptor tyrosine kinase, apolipoproteins, cholesterol, C-reactive protein (CRP), D-dimer, fibrinogen (Fib), glucose, insulin, interleukins, lipoproteins, myoglobin, N-terminal pro-B-type natriuretic peptide (BNP), tumor necrosis factor alpha (TNF-α) and troponins (Tns) on electrochemical transduction format. Identification of new specific CVD biomarkers, multiplex bioassay for the simultaneous determination of biomarkers, emergence of microfluidic biosensors, real-time analysis of biomarkers and point of care validation with high sensitivity and selectivity are the major challenges for future research., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This review (with 340 refs) focuses on methods for specific and sensitive detection of metabolites for diagnostic purposes, with particular emphasis on electrochemical nanomaterial-based sensors. It also covers novel candidate metabolites as potential biomarkers for diseases such as neurodegenerative diseases, autism spectrum disorder and hepatitis. Following an introduction into the field of metabolic biomarkers, a first major section classifies electrochemical biosensors according to the bioreceptor type (enzymatic, immuno, apta and peptide based sensors). A next section covers applications of nanomaterials in electrochemical biosensing (with subsections on the classification of nanomaterials, electrochemical approaches for signal generation and amplification using nanomaterials, and on nanomaterials as tags). A next large sections treats candidate metabolic biomarkers for diagnosis of diseases (in the context with metabolomics), with subsections on biomarkers for neurodegenerative diseases, autism spectrum disorder and hepatitis. The Conclusion addresses current challenges and future perspectives. Graphical abstract This review focuses on the recent developments in electrochemical biosensors based on the use of nanomaterials for the detection of metabolic biomarkers. It covers the critical metabolites for some diseases such as neurodegenerative diseases, autism spectrum disorder and hepatitis.

The sensitive quantification of Human Epidermal growth factor Receptor 2 (HER2), as a key prognostic tumor marker, plays a critical role in screening, early diagnosis and management of breast cancer. This paper describes a sandwich-type immunoassay with silver signal enhancement strategy for highly sensitive detection of HER2. For this purpose, the target capturing step was designed by functionalization of 3-aminopropyltrimethoxysilane coated magnetite nanoparticles with antibody (antiHER2/APTMS-Fe 3 O 4 ), as a platform bioconjugate (PB), and immobilized at a bare GCE. Then, in the presence of label-free immunosensor, the PB was covered by magnetic gold nanoparticles self-assembled with thiolated antibodies (antiHER2/Hyd@AuNPs-APTMS-Fe 3 O 4 ) containing chemically reduced silver ions, as a label bioconjugate (LB). Under optimum conditions, a linear relationship between the differential pulse voltammetric (DPV) stripping signal of silver and the logarithm of HER2 concentrations was obtained in the range of 5.0 × 10 -4 -50.0ngmL -1 (R 2 = 0.9906) with a detection limit of 2.0 × 10 -5 ngmL -1 . The effectiveness of this protocol was evaluated experimentally through employing of designed immunosensor for detection of the serum level of tumor marker. The good consistency of the results with those obtained by the enzyme-linked immunosorbent assay (ELISA) conventional method (p-value of < 0.05) showed that this immunosensor can be applied for the testing of HER2 in clinical samples of breast cancer patients., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The main advances in control and early diagnosis of cancer is greatly aided by low level detection of tumor markers in biological samples. Extensive efforts have been devoted to developing some ultrasensitive electrochemical biosensors for detection of cancer markers with high selectivity. These efforts include the development of the bioreceptors with high specificity and affinity, synthesis of novel signal amplifiers based on nanomaterials and the exploration of appropriate design strategies. Electrochemical measurement protocols are suitable for mass fabrication of miniaturized devices. They have a major role in the move towards rapid and simplified testing for point-of-care usage. This review discusses the remarkable advances of the last 6 years in the electrochemical affinity biosensors for determination of protein and glycoprotein tumor markers, with a particular focus on antibodies and aptamers as biorecognition probes., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Herein, for the first time a visible-light-driven photoelectrochemical (PEC) aptasensor for shrimp tropomyosin determination was fabricated by using graphitic carbon nitride (g-C 3 N 4 ) and titanium dioxide (TiO 2 ) as photoactive nanomaterials, ascorbic acid (AA) as electron donor and ruthenium (III) hexaammine (Ru(NH 3 ) 6 3+ ) as signal enhancer. The surface of an ITO electrode was first modified with g-C 3 N 4 , TiO 2 , and polyethyleneimine (PEI) and then the amine terminal aptamer TROP probe was attached to PEI by the use of glutaraldehyde (GA) as cross-linker. After that, Ru(NH 3 ) 6 3+ was adsorbed on aptamer to enhance the photocurrent signal. The principle of proposed PEC aptasensor is based on the formation of a selective complex between tropomyosin and immobilized aptamer TROP probe on the surface of ITO/g-C 3 N 4 -TiO 2 /PEI/aptamer TROP -Ru(NH3) 6 +3 . After the incubation of tropomyosin with TROP aptamer probe, the photocurrent signal decreased due to releasing adsorbed Ru(NH 3 ) 6 3+ on aptamer and preventing AA from scavenging photogenerated holes to the photoactive modified electrode. Under the optimized conditions, the fabricated PEC aptasensor was used for the determination of shrimp tropomyosin in the concentration range of 1-400ngmL -1 with a limit of detection of 0.23ngmL -1 . The proposed PEC aptasensor exhibited high selectivity, sensitivity, and good stability., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Recent developments in biotechnology offer the new methods for the sensitive detection of heavy metals based on the affinity and specificity of aptamers, as nucleic acid ligands selected from random sequence pools in vitro. Heavy metals have received considerable importance as the most toxic metallic pollutants which may cause serious environmental damages. They are classified as trace elements because of their presence in trace concentrations in various environmental matrices. Thus, the precise and sensitive methods to detect heavy metals are important to ensure human and environment safety. Aptamers as the biological probes, show high binding affinity which can often be directly translated into high detection sensitivity. On the other hand, high selectivity and stability make them possible to detect a wide range of targets, especially metallic ions. This review provides current progress of aptamers for environmental and biological monitoring of heavy metals using the nanomaterials mainly in two groups: (i) aptamer based biosensors (aptasensors) and (ii) aptamer based biosorbents (aptasorbents). The introduction of nanomaterials can efficiently increase the immobilization quantity of aptamers. Furthermore, they play an important role in the orientation and assembly density controlling of aptamers for the optimized recognition ability., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In this research, we demonstrated a flow injection amperometric sandwich-type aptasensor for the determination of human leukemic lymphoblasts (CCRF-CEM) based on poly(3,4-ethylenedioxythiophene) decorated with gold nanoparticles (PEDOT-Au nano ) as a nano platform to immobilize thiolated sgc8c aptamer and multiwall carbon nanotubes decorated with palladium nanoparticles/3,4,9,10-perylene tetracarboxylic acid (MWCNTs-Pd nano /PTCA) to fabricate catalytic labeled aptamer. In the proposed sensing strategy, the CCRF-CEM cancer cells were sandwiched between immobilized sgc8c aptamer on PEDOT-Au nano modified surface electrode and catalytic labeled sgc8c aptamer (MWCNTs-Pd nano /PTCA/aptamer). After that, the concentration of CCRF-CEM cancer cells was determined in presence of 0.1 mM hydrogen peroxide (H 2 O 2 ) as an electroactive component. The attached MWCNTs-Pd nano nanocomposites to CCRF-CEM cancer cells amplified the electrocatalytic reduction of H 2 O 2 and improved the sensitivity of the sensor to CCRF-CEM cancer cells. The MWCNT-Pd nano nanocomposite was characterized with transmission electron microscopy (TEM) and energy dispersive X-ray (EDX). The electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to confirm the stepwise changes in the electrochemical surface properties of the electrode. The proposed sandwich-type electrochemical aptasensor exhibited an excellent analytical performance for the detection of CCRF-CEM cancer cells ranging from 1.0 × 10 1 to 5.0 × 10 5  cells mL -1 . The limit of detection was 8 cells mL -1 . The proposed aptasensor showed high selectivity toward CCRF-CEM cancer cells. The proposed aptasensor was also applied to the determination of CCRF-CEM cancer cells in human serum samples., (Copyright © 2017 Elsevier B.V. All rights reserved.)

It is well known that cytochrome c (Cyt c) is a crucial death regulator that triggers programmed cell death, apoptosis. Here, we report on the successful application of an aptamer bioconjugated nanoclusters for the detection of apoptosis based on release of cytochrome c from mitochondria in lysates human embryonic kidney cells HEK293T. The aptamer-conjugated silver nanoclusters (Apt@AgNCs) were synthesized and immobilized on gold electrode for impedimetric detection of Cyt c over the range of 0.15-375nM. It was found that the presence of Ag(I) ions shell at the surface of prepared NCs could increase their affinity to the donor groups (COO - and some amine groups in its deprotonated form, NH 2 ) of cysteine attached on the gold electrode surface. The use of NCs, which play the role of conductive holders, established a simple immobilization procedure based on a low cost non-functionalized DNA sequence. To diminish the probable nonspecific impedance changes and decrease time of measurement, a rapid single frequency measurements (SFM) was assessed based on recording total impedance |Z| in different individual frequencies., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A very sensitive and convenient nanobiosensor based on fluorescence resonance energy transfer (FRET) was developed for the detection of a 22-mer oligonucleotides sequence in Human Papillomavirus 18 virus (HPV18) gene. For this purpose, water-soluble CdTe quantum dots (QDs) were synthesized and, subsequently, amino-modified 11-mer oligonucleotide as one of the two necessary probes was attached to QDs surface to form functional QDs-DNA conjugates. Right after addition of the QDs-DNA and a second Cyanine5 (Cy5)-labeled 11-mer oligonucleotide probe to the DNA target solution, the sandwiched hybrids were formed. The resulting hybridization brings the Cy5 fluorophore as the acceptor to close proximity of the QDs as donor, so that an effective transfer of energy from the excited QDs to the Cy5 probe would occur via FRET processing. The fluorescence intensity of Cy5 found to linearly enhance by increasing the DNA target concentration from 1.0 to 50.0nM, with a detection limit of 0.2nM. This homogeneous DNA detection method does not require excessive washing and separation steps of un-hybridized DNA, due to the fact that no FRET can be observed when the probes are not ligated. Finally, feasibility and selectivity of the proposed one-spot DNA detection nanobiosensor were investigated by analysis of derived nucleotides from HPV18 and mismatched sequences., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A "signal off" voltammetric aptasensor was developed for the sensitive and selective detection of ultra-low levels of adenosine triphosphate (ATP). For this purpose, a new strategy based on the principle of recognition-induced switching of aptamers from DNA/DNA duplex to DNA/target complex was designed using thioglycolic acid (TGA)-capped CdTe quantum dots (QDs) as the signal amplifying nano-platforms. Owing to the small size, high surface-to-volume ratio and good conductivity, quantum dots were immobilized on the electrode surface for signal amplification. In this work, methylene blue (MB) adsorbed to DNA was used as a sensitive redox reporter. The intensity of voltammetric signal of MB was found to decrease linearly upon ATP addition over a concentration range of 0.1nM to 1.6μM with a correlation coefficient of 0.9924. Under optimized conditions, the aptasensor was able to selectively detect ATP with a limit of detection of 45pM at 3σ. The results also demonstrated that the QDs-based amplification strategy could be feasible for ATP assay and presented a potential universal method for other small biomolecular aptasensors., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In this study, preparation of novel pH-sensitive N-doped carbon dots (NCDs) using glucose and urea is reported. The prepared NCDs present strong excitation-dependent fluorescence changes towards the pH that is a new behavior from these nanomaterials. By taking advantage of this unique behavior, two separated ratiometric pH sensors using emission spectra of the NCDs for both acidic (pH 2.0 to 8.0) and basic (pH 7.0 to 14.0) ranges of pH are constructed. Additionally, by considering the entire Excitation-Emission Matrix (EEM) of NCDs as analytical signal and using a suitable multivariate calibration method, a broad range of pH from 2.0 to 14.0 was well calibrated. The multivariate calibration method was independent from the concentration of NCDs and resulted in a very low average prediction error of 0.067 pH units. No changes in the predicted pH under UV irradiation (for 3 h) and at high ionic strength (up to 2 M NaCl) indicated the high stability of this pH nanosensor. The practicality of this pH nanosensor for pH determination in real water samples was validated with good accuracy and repeatability., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A simple and sensitive method for the detection of DNA hybridization in a homogeneous format was developed, using bis(8-hydroxyquinoline-5-solphonate)cerium(III) chloride (Ce(QS)2Cl) as a novel fluorescent probe. The method is based on fluorescence quenching by gold nanoparticles used as both nanoscafolds for the immobilization of the probe DNA sequence, which is related to Alicyclobacillus acidophilus strain TA-67 16S ribosomal RNA, and nanoquenchers of the Ce(QS)2Cl probe. The probe DNA-functionalized GNPs were synthesized by derivatizing the colloidal gold nanoparticles solution with 3-thiolated 16-base oligonucleotides. Addition of sequence-specific target DNAs (16 bases) into the mixture containing probe DNA-functionalized GNPs and fluorescent probe lead to the quenching of Ce(QS)2Cl fluorescence at 360 nm (λex=270 nm), due to DNA hybridization, the resulting quenched intensity being proportional to the concentration of target DNA. Under optimal conditions of pH 7.4 and Ce(QS)2Cl concentration of 1.0 × 10(-7) M, the linear dynamic range found to be 1.0 × 10(-10)-3.0 × 10(-8) M DNA, with a limit of detection of 7.0 × 10(-11) M. The interaction mechanism for the binding of Ce(QS)2Cl to DNA was studied in detail, and results proved that the interaction mode between Ce(QS)2Cl and DNA is groove binding, with a binding constant of 1.0 × 10(5) M(-1)., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Herein, a label free electrochemical aptasensor based on ordered mesoporous carbon-gold nanocomposite modified screen printed electrode has been fabricated for the detection of vascular endothelial growth factor (VEGF165) as a tumor marker. The electrochemical behavior of prepared biosensor was investigated by cyclic voltammetry and electrochemical impedance spectroscopy. The principle of operation of the proposed aptasensor is based on the changes in the interfacial properties of the electrode due to interaction of the immobilized antiVEGF165 aptamer at the electrode surface with VEGF165 tumor marker in the sample solution, which results in a change in the interfacial charge transfer resistance as detected by electrochemical impedance spectroscopy. The calibration curve for VEGF165 determination was linear over 10.0-300.0 pg mL(-1) with a limit of detection (3σ/S) of 1.0 pg mL(-1). The prepared aptasensor exhibited high sensitivity and good selectivity and reproducibility. The aptasensor was successfully applied to the determination of VEGF165 in serum sample of a lung cancer patient., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Herein, an easy and cost-effective approach to the immobilization of probe was performed. The amino modified salmonella ssDNA probe sequence was covalently linked with carboxylic group on the surface of nanoporous glassy carbon electrode to prepare the DNA biosensor. The differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) techniques were used for the determination of salmonella DNA in the concentration ranges of 10-400pM and 1-400pM with limits of detection of 2.1pM and 0.15pM, respectively., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A label free immunosensor was designed for ultra-detection of human epidermal growth factor receptor 2 (HER2) in real samples using a differential pulse voltammetry (DPV) method. In a separate process, antiHER2 antibodies were attached to iron oxide nanoparticles (Fe3O4 NPs) to form stable bioconjugates which were later laid over the gold electrode surface. In this way, by the advantage of their long terminals, the bioconjugates provided the most possible space for the immuno-reaction between biomolecules. Under optimal conditions, the immunosensor was responsive to HER2 concentrations over the ranges of 0.01-10 ng mL(-1) and 10-100 ng mL(-1) linearly and benefited from a satisfactory detection limit as low as 0.995 pg mL(-1) and a favorable sensitivity as sharp as 5.921 μA mL ng(-1). The reliability of the method in clinical analysis was proved by successful quantization of HER2 levels in serum samples obtained from patients. Furthermore, the precision and the stability of the method were evaluated and verified to be acceptable in immunoassay studies.

A highly sensitive ionic liquid-multiwall carbon nanotube based impedimetric immunosensor modified with gold nanoparticles (GNPs) was developed for the determination of human serum albumin (HSA). The antigen and antibody models used were HSA and activated anti-HSA, respectively. GNPs were electrodeposited on the multiwall based carbon ionic liquid electrode (MW-CILE) surface and then colloidal GNPs were coated through the thiol groups of 1,6-hexanedithiol (HDT) monolayer as a cross linker. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were employed for characterization of the various layers coated onto the electrode. The electron transfer resistance (Ret) of the antibody-modified electrode changed linearly with the concentration of HSA. The linear range and limit of detection were 0.1-100μgmL(-1) and 15.4ngmL(-1), respectively. The sensitivity and specificity of the immunosensor were validated using human urine and human serum samples. The results showed that the prepared immunosensor is a useful tool for screening trace amounts of HSA in the biological fluids of proteinuria and diabetics patients, in clinical laboratories., (Copyright © 2014 Elsevier B.V. All rights reserved.)

This work reports a novel nanobiosensor based on a thioglycolic acid (TGA)-capped CdTe quantum dot-laccase (Lac) enzyme system for sensitive detection of dopamine (DA). The enzyme used catalyzes the oxidation of DA to dopamine-o-quinone (DOQ), which can selectively quench the strong luminescence of CdTe nanocrystals at neutral pH. The relationship between luminescence intensity of CdTe nanocrystals and DA concentration is nicely described by the Stern-Volmer equation. At an optimum pH of 7.4, the proposed sensor gives a linear calibration over a DA concentration range of 0.3 to 100 μM, with a limit of detection of 0.16 μM and a response time of 2 min. The relative standard deviation for seven replicate determinations of 6.0 μM of DA was found to be 3.7%. The sensor was successfully applied to the determination of DA in a blood plasma sample and in a DA injection formulation.

A novel glucose biosensor based on the chemiluminescence (CL) detection of enzymatically generated H(2)O(2) was constructed by the effective immobilization of glucose oxidase (GOD)/carbon-nanotubes (CNTs)/gold nanoparticles (GNPs) in nafion film on graphite support. The influences of various experimental parameters such as solution pH, the action time of the enzyme, interferents and the concentration of CL reagents were investigated. Carbon nanotubes and gold nanoparticles offer excellent catalytic activity toward hydrogen peroxide generation in enzymatic reaction between glucose oxidase and glucose, which would enable sensitive determination of glucose. Under the optimum condition, the linear response range of glucose was found to be 2.25 × 10(-6) to 1.75 × 10(-4 ) mol L(-1), and the detection limit (defined as the concentration that could be detected at the signal-to-noise ratio of 3) was 1.00 × 10(-6) mol L(-1). The CL biosensor exhibited good storage stability, i.e., 80% of its initial response was retained after 10 days storage at pH 7.0. The present CL biosensor has been used to determine the glucose concentrations in real serum and urine samples with satisfactory results., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A robust and effective nanocomposite film-glassy carbon modified electrode based on multi-walled carbon nanotubes and a room temperature ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate was prepared by a layer-by-layer self-assembly method. The fabricated modified electrode was used as a novel impedimetric catalase nanobiosensor for the determination of H(2)O(2). Direct electron transfer and electrocatalysis of catalase were fully investigated. The results suggested that catalase could be firmly adsorbed at the modified electrode. A pair of quasi-reversible redox peaks of catalase was observed in a 0.20 M degassed phosphate buffer solution of pH 7.0. The nanocomposite film showed a pronounced increase in direct electron transfer between catalase and the electrode. The immobilized catalase exhibited an excellent electrocatalytic activity towards the reduction of H(2)O(2). The electrochemical impedance spectroscopy measurements revealed that the charge transfer resistance decreases significantly after enzymatic reaction with hydrogen peroxide, so that the prepared modified electrode can be used for the detection of ultra traces of H(2)O(2) (5-1700 nM)., (Copyright © 2011 Elsevier B.V. All rights reserved.)

An electrochemical DNA nano-biosensor is prepared by immobilization of double stranded DNA (dsDNA) onto a mixed self-assembled monolayer (SAM) composed of azide- and hydroxyl-terminated thiols. The SAMs- and dsDNA-modified gold electrodes were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The interaction of spermidine with DNA was studied by differential pulse voltammetry at the DNA-modified electrode. The decrease in the guanine oxidation peak current was used to study the interaction. The binding constant (K), obtained by differential pulse voltammetry, was 1.85 x 10(5)M(-1). A linear dependence of the guanine peak currents was observed in the range of 1.6-70.4 microM spermidine, with a detection limit of 0.72 microM and r=0.994 by using differential pulse voltammetry.

Electrochemical impedance spectroscopy (EIS) as a powerful, non-invasive and informative technique was used to obtain important information about kinetics of doping process in conducting polymers. Polypyrrole (PPy) and its derivatives can form conducting polymer films which represent excellent charge transfer behaviors during doping processes. It can also have a wide range of applications in bioelectrochemistry. In the present study the conducting polymer of alpha-carboxy pyrrole (alpha-COOH-PPy), appended onto the underlying film of PPy, was prepared by electrochemical methods and its behavior was analyzed using EIS. From highly accurate fitting of impedance results it was found that the charging mechanism is governed by the diffusion process. In addition, the impedance analyses provided values for the bulk polymer parameters including diffusion coefficient (D), equilibrium capacitance (C(0)) and diffusion resistance (R(0)). The surface morphology of the polymeric film was characterized using scanning electron microscopy (SEM). The film was then used to immobilize the cytochrome C (cyt-C) and to perform its electrochemical studies. The modified cyt-C/alpha-COOH-PPy electrode was used for electrocatalytic reduction of H(2)O(2) in solution and its viability as a new impedimetric biosensor was examined. Based on the calibration curve obtained for the proposed impedimetric biosensor, the limit of detection and relative standard deviation were evaluated as 0.25 micromolL(-1) and 7%, respectively. Finally, the prolonged stability test was performed and high stability and reproducibility of the new biosensor was confirmed.

A novel diclofenac ion-selective electrode is prepared, characterized and used in pharmaceutical analysis. The diclofenac complex with hexadecylpyridinium bromide is obtained in situ by soaking the PVC-membranes in a 1x10(-2) M diclofenac solution. Among four different solvent mediators tested, dibutyl phthalate (DBP) exhibited a proper behavior including Nernstian slope of the calibration curve, fast response time and good reproducibility of the emf values. The electrode exhibits a Nernstian slope of -59+/-1 mV decade(-1) for diclofenac in the concentration range 1.0x10(-5) to 1.0x10(-2) M with a limit of detection of 4.0x10(-6) M. The electrode displays a good selectivity for diclofenac with respect to a number of common inorganic and organic species. It can be used in a pH range of 6.0-9.0. The membrane sensor was successfully applied to the determination of diclofenac in its tablets as well as for its recovery from blood serum and urine samples.